High-risk types of human papillomaviruses (HPV) are small, non-enveloped DNA viruses etiologically associated with 5% of human cancers, including essentially all cervical cancer, most other anogenital cancer, and an increasing fraction of oropharyngeal cancer. Vaccines have been developed that target some types of HPV, but the great majority of people will remain unvaccinated for the foreseeable future. Little is known about the intracellular events occurring during HPV entry into cells. To understand this important process in molecular detail, we performed a genome-wide RNA interference screen for genes required for infection of cervical cancer cells by HPV16 pseudovirus. The screen was technically robust and successfully identified several factors known to be required for HPV infection. In addition, the screen identified numerous novel essential factors, including several factors involved in retrograde transport to the Golgi apparatus. Prominent validated hits encode components of the retromer, a molecular machine involved in transport of cargo from endosomes to Golgi. In addition, incoming, partially disassembled HPV16 capsids localize to the trans-Golgi network (TGN) in a retromer-dependent fashion, and components of the retromer are in a physical complex with HPV capsid proteins in infected cells. Finally, a small molecular inhibitor of retrograde transport also specifically inhibits infection. Notably, the retromer has nt been previously implicated in entry of any virus. On the basis of these exciting discoveries, we hypothesize that retromer and associated pathways directly transport HPV into the TGN during virus entry. We propose experiments to test this hypothesis and identify the intracellular trafficking pathway(s) utilized by HPV16. In aim 1, we will conduct genetic and cell localization experiments to explore the role of the endosome-to-Golgi retromer transport system during HPV infection and identify additional essential factors. In aim 2, we will conduct biochemical and imaging experiments to identify and characterize proteins that associate with incoming HPV16 capsids, with a focus on retrograde pathway components. In aim 3, we will conduct mutational and other analyses to test our hypothesis that retromer supports HPV infection by participating in the transfer of HPV constituents to the TGN. Overall, these experiments will elucidate the mechanistic details of HPV entry, provide new insights into fundamental cell biology, and suggest novel anti-viral strategies.